Conditional Gene Knockout by SMOC

Introduction to Conditional Gene Knockout

• Overview: Explaining the concept of conditional knockout (CKO) models, which enable gene knockout in a specific temporal or spatial pattern, allowing for more focused and accurate research.

Tab 1 CKO
 

• Site-Specific Recombinase Systems: Mainly achieved through systems like Cre-LoxP, FLP-Frt, and Dre-Rox, with Cre-LoxP being the most frequently used.
• Flox Mouse Models: A LoxP sequence is placed at each end of a DNA sequence to be deleted, generating a Flox (flanked by LoxP) mouse. Flox mice are then crossed with Cre-expressing mice for tissue- or cell-specific gene knockout.

• Reduced Lethality Risks: Flox mice may be less likely to develop embryonic or neonatal lethality compared to conventional knockout models.
• Flexibility: By crossing with different Cre-expressing mice, offsprings can be generated in which the target gene is inactivated at any developmental stage or in any specific cell type.

Timeframe for CRISPR Gene Editing

• It typically takes 6-9 months to generate a conditional gene knockout mouse model using CRISPR gene editing technology.

Tab 2 Systems

Cre-lox System

• Efficiency: Characterized by simple manipulation and high efficiency, the Cre-lox system is a powerful tool for gene editing.
• Applications: Allows for time-specific knockout and knock-in of target genes, thereby enabling spatiotemporal regulation of genes .

Recombination Events in Cre-lox System

• Deletion: Occurs if the two loxP sites are in the same orientation.
• Inversion: Happens if the loxP sites are in opposite orientations.
• Translocation: If there's a floxed donor sequence, it can be swapped with the original sequence.

Cre Recombination Mechanism

• Figure 2: Illustrating the structure of loxP sites and the Cre recombinase-mediated cassette exchange, inversion, and excision.

Steps in Establishing Conditional Gene Targeting

• Cre-expressing Mouse Strain: Driven by specific promoters, Cre recombinase can function in specific cells, tissues, or the whole body.
• Flox Mouse Strain: Established by flanking the target gene with lox sites. The conditional gene targeting strain is achieved by crossing Cre-expressing and Flox mouse strains.

Conditional Gene Activation and Knock-Out

• Gene Activation: Utilizing the Lox-Stop-Lox (LSL) stop cassette for cell type-specific gene activation.

• Gene Knock-Out: Achieving conditional gene knockout by flanking the target gene with lox sites in the same orientation.

Inducible Cre-lox Systems

Tab 3  ES cell targeting